( 2003 )
Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17.
PMID : 12687298 : DOI : 10.1007/s00253-003-1250-6
A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.
( 2002 )
Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences.
PMID : 12489780 : DOI : 10.1139/w02-089
The use of hsp60 gene sequences for phylogenetic study and identification of pathogenic marine vibrios was investigated. A 600-bp partial hsp60 gene was amplified by PCR and sequenced from 29 strains representing 15 Vibrio species within the family Vibrionaceae. Sequence comparison of the amplified partial hsp60 gene revealed 71-82% sequence identity among different Vibrio species and 96-100% sequence identity among epidemiologically distinct strains with the same species designation. This degree of discrimination allows unambiguous differentiation of all Vibrio species included in the current study from each other, as well as from Aeromonas hydrophila and Plesiomonas shigelloides, which are often misidentified as Vibrio species by conventional biochemical methods. Based on the hsp60 gene sequences, two previously unidentified shrimp isolates were found to be more closely related to Vibrio alginolyticus (93-94% sequence identity) than to Vibrio parahaemolyticus (89% sequence identity), whereas 16S rRNA gene analysis was unable to differentiate among these closely related species (95-97% sequence identity). Our results indicate that the hsp60 gene may be a useful alternative target for phylogenetic analysis and species identification of marine Vibrios to complement more conventional identification systems.
( 2002 )
Purification, characterization and molecular cloning of Vibrio fluvialis hemolysin.
PMID : 12479411 : DOI : 10.1016/s1570-9639(02)00407-7
Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q. N-terminal amino acid sequences of the purified VFH were determined. The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH. Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations. Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay. Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa. Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids. Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.
( 2002 )
An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning.
PMID : 12220989 :
An exocellular metalloprotease produced by Vibrio fluvialis, an enteropathogenic vibrio, was purified and characterized. The metalloprotease (V. fluvialis protease [VFP]) was found to have very similar characteristics to V. vulnificus protease, including a molecular mass of 45kDa, sensitivity to chelating agents or competitive inhibitors for thermolysin-like metalloproteases, and the substrate specificity. The structural gene for VFP was also cloned, and its nucleotide sequence was determined. The deduced amino acid sequence confirmed that VFP was a member of the thermolysin family. VFP, like V. vulnificus protease, showed the haemagglutinating, permeability-enhancing and haemorrhagic activities in addition to the proteolytic activity toward oligopeptide, casein or elastin.
( 2000 )
A region of the transmembrane regulatory protein ToxR that tethers the transcriptional activation domain to the cytoplasmic membrane displays wide divergence among Vibrio species.
PMID : 10629204 : DOI : 10.1128/jb.182.2.526-528.2000 PMC : PMC94307
The virulence regulatory protein ToxR of Vibrio cholerae is unique in that it contains a cytoplasmic DNA-binding-transcriptional activation domain, a transmembrane domain, and a periplasmic domain. Although ToxR and other transmembrane transcriptional activators have been discovered in other bacteria, little is known about their mechanism of activation. Utilizing degenerate oligonucleotides and PCR, we have amplified internal toxR gene sequences from seven Vibrio and Photobacterium species and subspecies, demonstrating that toxR is an ancestral gene of the family Vibrionaceae. Sequence alignment of all available ToxR amino acid sequences revealed a region between the transcriptional activation and transmembrane domains that displays wide divergence among Vibrio species. We hypothesize that this region merely tethers the transcriptional activation domain to the cytoplasmic membrane and thus can tolerate wide divergence and multiple insertions and deletions. The divergence in the tether region at the nucleotide level may provide a useful tool for the distinction of Vibrio and Photobacterium species.
( N/A )
Studies on the region involved in the transport activity of Escherichia coli TolC by chimeric protein analysis.
PMID : 17350794 : DOI : 10.1016/j.micpath.2007.01.006
Gram-negative bacteria possess the outer membrane protein TolC which acts as an exit duct across the outer membrane. However, the region involved in the transport activity of TolC has remained unclear. We analyzed this region by creating chimeric TolCs. First, we expressed the genes for TolCs of Vibrio parahaemolyticus (vp-tolC) and Salmonella typhimurium (sal-tolC) in Escherichia coli. The levels of sequence identity in the mature region of VP-TolC/EC-TolC and Sal-TolC/EC-TolC with maximum matching are 43% and 90%, respectively. We found that the transport activity of VP-TolC was weak compared with that of TolC of E. coli (EC-TolC) although the transport activity of Sal-TolC was similar to that of EC-TolC. A comparison of the sequence of the three tolCs showed that the sequence around the periplasmic region covering Asn-188 to Lys-214 of EC-TolC is lowly identical to that of VP-TolC although the region of EC-TolC is almost identical to that of Sal-TolC. We think, therefore, that the region covering Asn-188 to Lys-214 of EC-TolC may have an important role to express its transport activity in E. coli. To examine the possibility, we divided the region of EC-TolC into three and exchanged the gene for each portion with that of vp-tolC. These mutant ec-tolCs were expressed in E. coli and the activity of each chimeric TolC was measured. The results showed that the portion covering Val-198 to Lys-214 of EC-TolC is deeply involved in the transport activity.
( 2007 )
The SXT/R391 family of integrative conjugative elements is composed of two exclusion groups.
PMID : 17307849 : DOI : 10.1128/JB.01902-06 PMC : PMC1855829
Conjugative elements often encode entry exclusion systems that convert host cells into poor recipients for identical or similar elements. The diversity of exclusion systems within families of conjugative elements has received little attention. We report here the most comprehensive study to date of the diversity of exclusion determinants within a single family of conjugative elements. Unexpectedly, our analyses indicate that there are only two exclusion groups among the diverse members of the SXT/R391 family of integrative conjugative elements.
( 2007 )
The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species.
PMID : 17207598 : DOI : 10.1016/j.syapm.2006.11.004
The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.
( 2007 )
Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus.
PMID : 17172518 : DOI : 10.1099/jmm.0.46759-0
The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization.
( 2007 )
Identification of Vibrio isolates by a multiplex PCR assay and rpoB sequence determination.
PMID : 17093013 : DOI : 10.1128/JCM.01544-06 PMC : PMC1828960
Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus-account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.
( 2006 )
Mechanism of drug resistance in clonally related clinical isolates of Vibrio fluvialis isolated in Kolkata, India.
PMID : 16801422 : DOI : 10.1128/AAC.01561-05 PMC : PMC1489780
The molecular mechanisms of drug resistance in 19 strains of Vibrio fluvialis isolated from 1998 to 2002 in Kolkata, India, were investigated. Class 1 integrons were detected in eight strains, and four strains were found to carry SXT integrases. In the presence of carbonyl cyanide m-chlorophenylhydrazone or reserpine, all nalidixic acid- and ciprofloxacin-resistant strains became sensitive, suggesting that drug efflux plays a major role in quinolone resistance in V. fluvialis. It was further seen that strains which had MICs of > 25 microg/ml for nalidixic acid had a sense mutation (Ser to Ile) at position 83 of the quinolone resistance-determining region of gyrA. All except one of the integron- and SXT integrase-bearing strains belonged to the same ribotype.
( 2005 )
Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis.
PMID : 16151093 : DOI : 10.1128/AEM.71.9.5107-5115.2005 PMC : PMC1214639
We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.
( 2005 )
Identification of an iron-regulated hemin-binding outer membrane protein, HupO, in Vibrio fluvialis: effects on hemolytic activity and the oxidative stress response.
PMID : 15664910 : DOI : 10.1128/IAI.73.2.722-729.2005 PMC : PMC546946
In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.
( 2005 )
A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis.
PMID : 15621444 : DOI : 10.1016/j.femsle.2004.11.012
Vibrio fluvialis strain H-08942 was isolated from an infant aged 6 months who was suffering from cholera-like diarrhea in India. This strain showed the typical multidrug-resistance phenotype of an SXT element. It was resistant to sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm) and streptomycin (Sm), in addition to other antibiotics such as ampicillin (Am), furazolidone (Fz), nalidixic acid (Na), and gentamicin (Gm). The SXT element is a Vibrio cholerae-derived integrative and conjugative element (ICE) that has also been referred to as a conjugative transposon. Our goal was to find a relationship between these resistant phenotypes and the presence of the SXT element in this unique strain. By using PCR, we detected the antibiotic resistance genes, the integrase gene and the attP attachment site of SXT element. Cloning and DNA sequencing results showed that both the SXT integrase gene and its attP site of V. fluvialis were similar but not identical to those of V. cholerae. The SXT integrase gene of V. fluvialis has a 99% identity to that of V. cholerae, and the attP site of SXT of V. fluvialis is variant and shorter (641 bp) than that of V. cholerae (785 bp). It was possible for the SXT of V. fluvialis to be transferred by conjugation to a laboratory strain of Escherichia coli. Here, we report the detection of a variant SXT element in species other than V. cholerae, with molecular characterization and analysis of its integrase gene and its attP site.
( 2004 )
Identification of oligopeptide permease (opp) gene cluster in Vibrio fluvialis and characterization of biofilm production by oppA knockout mutation.
PMID : 15500975 : DOI : 10.1016/j.femsle.2004.09.007
Oligopeptides play important roles in bacterial nutrition and signaling. The oligopeptide permease (opp) gene cluster was cloned from Vibrio fluvialis. The V. fluvialis opp operon encodes five proteins: OppA, B, C, D and F. The deduced amino acid sequence of these proteins showed high similarity with those from other Gram-negative bacteria. To investigate whether OppA is involved in biofilm production, an oppA knockout mutant was constructed by homologous recombination. The oppA mutant produced more abundant biofilm than the wild type in BHI medium. When both strains were grown in minimal medium, we could not detect biofilm formation. However, it was found that the biofilm productivity of the oppA mutant was two folds greater than that of the wild type in minimal medium containing peptone or tryptone. This variation in biofilm production was demonstrated by scanning electron microscopy (SEM). In minimal medium containing C-sources, both strains produced some biofilm without significant difference in the biofilm productivity. Complementation of oppA gene with the plasmid pOAC2, which contains oppA ORF plus promoter regions, was sufficient to restore growth rate and biofilm to the wild type. These results suggest that the OppA protein is involved in uptake of peptides and affects biofilm productivity.
( 2004 )
Use of recA as an alternative phylogenetic marker in the family Vibrionaceae.
PMID : 15143042 : DOI : 10.1099/ijs.0.02963-0
This study analysed the usefulness of recA gene sequences as an alternative phylogenetic and/or identification marker for vibrios. The recA sequences suggest that the genus Vibrio is polyphyletic. The high heterogeneity observed within vibrios was congruent with former polyphasic taxonomic studies on this group. Photobacterium species clustered together and apparently nested within vibrios, while Grimontia hollisae was apart from other vibrios. Within the vibrios, Vibrio cholerae and Vibrio mimicus clustered apart from the other genus members. Vibrio harveyi- and Vibrio splendidus-related species formed compact separated groups. On the other hand, species related to Vibrio tubiashii appeared scattered in the phylogenetic tree. The pairs Vibrio coralliilyticus and Vibrio neptunius, Vibrio nereis and Vibrio xuii and V. tubiashii and Vibrio brasiliensis clustered completely apart from each other. There was a correlation of 0.58 between recA and 16S rDNA pairwise similarities. Strains of the same species have at least 94 % recA sequence similarity. recA gene sequences are much more discriminatory than 16S rDNA. For 16S rDNA similarity values above 98 % there was a wide range of recA similarities, from 83 to 99 %.
( 2004 )
New aminoglycoside acetyltransferase gene, aac(3)-Id, in a class 1 integron from a multiresistant strain of Vibrio fluvialis isolated from an infant aged 6 months.
PMID : 15117923 : DOI : 10.1093/jac/dkh221
To characterize the molecular basis of antibiotic resistance in a multidrug-resistant clinical isolate of Vibrio fluvialis H-08942. V. fluvialis H-08942 was isolated from a hospitalized infant aged 6 months suffering from cholera-like diarrhoea in India in 2002. The broth microdilution method was used to determine the MICs of a range of antibiotics for this strain. PCR, DNA sequencing, Southern hybridization, cloning and expression were used to characterize the molecular basis of antibiotic resistances. V. fluvialis H-08942 showed resistance to chloramphenicol, streptomycin, spectinomycin, co-trimoxazole, ampicillin, furazolidone, nalidixic acid and gentamicin. A class 1 integron that contains a novel aminoglycoside acetyltransferase gene, aac(3)-Id, and aminoglycoside adenyltransferase gene, aadA7, was characterized. The aac(3)-Id gene product was found to share 50%, 45% and 44% identity to AAC(3)-Ic, AAC(3)-Ia, and AAC(3)-Ib, respectively. Both aac(3)-Id and aadA7 genes were cloned and expressed in Escherichia coli. Phylogenetic analysis suggested that the aac(3)-Id represents a fourth evolutionary lineage in the aminoglycoside acetyltransferase genes. Southern hybridization showed that this integron is located in the chromosome. In this study we identified a new type of aminoglycoside acetyltransferase gene, aac(3)-Id. In addition, this is the first report of identification of antibiotic resistance genes and a class 1 integron in V. fluvialis.
( 2011 )
Multiplex PCR assay for identification of three major pathogenic Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus.
PMID : 21530641 : DOI : 10.1016/j.mcp.2011.04.004
A multiplex PCR assay was developed based on atpA-sequence diversification for molecular identification of 3 major pathogenic Vibrio species: Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. It specifically identified them from among 133 strains of various Vibrio species and other genera, and was applicable for testing seawater, suggesting its usefulness.
( 2011 )
Detection of quorum sensing signal molecules in the family Vibrionaceae.
PMID : 21395950 : DOI : 10.1111/j.1365-2672.2011.04998.x
The aim of this study was to detect the production of three kinds of quorum sensing (QS) signal molecules, i.e. the N-acyl-homoserine lactone (AHL), the autoinducer-2 (AI-2) and the cholerae autoinducer-1-like (CAI-1-like) molecules in 25 Vibrionaceae strains. The QS signal molecules in 25 Vibrionaceae strains were detected with different biosensors. Except Salinivibrio costicola VIB288 and Vibrio natriegens VIB299, all the other 23 Vibrionaceae strains could produce one or more kinds of detectable QS signal molecules. Twenty-one of the 25 strains were found to produce AHL signal molecules by using Vibrio harveyi JMH612 and Agrobacterium tumefaciens KYC55 (pJZ372; pJZ384; pJZ410) as biosensors. The AHL fingerprints of eight strains were detected by thin-layer chromatography with Ag. tumefaciens KYC55, and two of them, i.e. V. mediterranei VIB296 and Aliivibrio logei VIB414 had a high diversity of AHLs. Twenty of the 25 strains were found to have the AI-2 activity, and the luxS gene sequences in 18 strains were proved to be conserved by PCR amplification and sequencing. Only six (five Vibrio strains and A. logei VIB414) of the 25 strains possessed the CAI-1-like activity. A. logei VIB414, V. campbellii VIB285, V. furnissii VIB293, V. pomeroyi LMG20537 and two V. harveyi strains VIB571 and VIB645 were found to produce all the three kinds of QS signal molecules. The results indicated that the QS signal molecules, especially AHL and AI-2 molecules, were widespread in the family Vibrionaceae. In response to a variety of environmental conditions and selection forces, the family Vibrionaceae produced QS signal molecules with great diversity and complexity. The knowledge we obtained from this study will be useful for further research on the roles of different QS signal molecules in this family.
( 2010 )
Integrating conjugative elements of the SXT/R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands.
PMID : 20807202 : DOI : 10.1111/j.1365-2958.2010.07364.x
In vibrios and enterobacteria lateral gene transfer is often facilitated by integrating conjugative elements (ICEs) of the SXT/R391 family. SXT/R391 ICEs integrate by site-specific recombination into prfC and transfer by conjugation, a process that is initiated at a specific locus called the origin of transfer (oriT(SXT)). We identified genomic islands (GIs) harbouring a sequence that shares >63% identity with oriT(SXT) in three species of Vibrio. Unlike SXT/R391 ICEs, these GIs are integrated into a gene coding for a putative stress-induced protein and do not appear to carry any gene coding for a conjugative machinery or for mobilization proteins. Our results show that SXT/R391 ICEs trigger the excision and mediate the conjugative transfer in trans of the three Vibrio GIs at high frequency. GIs' excision is independent of the ICE-encoded recombinase and is controlled by the ICE-encoded transcriptional activator SetCD, which is expressed during the host SOS response. Both mobI and traI, two ICE-borne genes involved in oriT recognition, are essential for GIs' transfer. We also found that SXT/R391 ICEs mobilize in trans over 1 Mb of chromosomal DNA located 5' of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.
( 2009 )
Comparative ICE genomics: insights into the evolution of the SXT/R391 family of ICEs.
PMID : 20041216 : DOI : 10.1371/journal.pgen.1000786 PMC : PMC2791158
Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements.
( 2009 )
Mechanism of drug resistance in a clinical isolate of Vibrio fluvialis: involvement of multiple plasmids and integrons.
PMID : 19427174 : DOI : 10.1016/j.ijantimicag.2009.03.020
The role of mobile genetic elements in imparting multiple drug resistance to a clinical isolate of Vibrio fluvialis (BD146) was investigated. This isolate showed complete or intermediate resistance to all of the 14 antibiotics tested. Polymerase chain reaction (PCR) revealed the presence of a class 1 integron and the absence of the SXT element in this isolate. The strain harboured a 7.5 kb plasmid and a very low copy number plasmid of unknown molecular size. Transformation of Escherichia coli with plasmid(s) from BD146 generated two kinds of transformants, one that harboured both of these plasmids and the other that harboured only the low copy number plasmid. PCR and antibiogram analysis indicated the association of the class 1 integron with the low copy number plasmid, which also conferred all the transferable resistance traits except trimethoprim to the parent strain. A BLAST search with the sequence of the 7.5kb plasmid showed that it was 99% identical to plasmid pVN84 from Vibrio cholerae O1 in Vietnam, indicating that these two plasmids are probably one and the same. To the best of our knowledge, this is the first report of horizontal transfer of a plasmid between V. fluvialis and V. cholerae.
( 2007 )
Phylogenetic analysis of vibrios and related species by means of atpA gene sequences.
PMID : 17978204 : DOI : 10.1099/ijs.0.65223-0
We investigated the use of atpA gene sequences as alternative phylogenetic and identification markers for vibrios. A fragment of 1322 bp (corresponding to approximately 88% of the coding region) was analysed in 151 strains of vibrios. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. For instance, the Vibrio cholerae, Vibrio halioticoli, Vibrio harveyi and Vibrio splendidus species groups appeared in the atpA gene phylogenetic analyses, suggesting that these groups may be considered as separate genera within the current Vibrio genus. Overall, atpA gene sequences appeared to be more discriminatory for species differentiation than 16S rRNA gene sequences. 16S rRNA gene sequence similarities above 97% corresponded to atpA gene sequences similarities above 80%. The intraspecies variation in the atpA gene sequence was about 99% sequence similarity. The results showed clearly that atpA gene sequences are a suitable alternative for the identification and phylogenetic study of vibrios.
( 2017 )
Functional Characterization and Conditional Regulation of the Type VI Secretion System in Vibrio fluvialis.
PMID : 28424671 : DOI : 10.3389/fmicb.2017.00528 PMC : PMC5371669
Vibrio fluvialis is an emerging foodborne pathogen of increasing public health concern. The mechanism(s) that contribute to the bacterial survival and disease are still poorly understood. In other bacterial species, type VI secretion systems (T6SSs) are known to contribute to bacterial pathogenicity by exerting toxic effects on host cells or competing bacterial species. In this study, we characterized the genetic organization and prevalence of two T6SS gene clusters (VflT6SS1 and VflT6SS2) in V. fluvialis. VflT6SS2 harbors three "orphan" hcp-vgrG modules and was more prevalent than VflT6SS1 in our isolates. We showed that VflT6SS2 is functionally active under low (25�XC) and warm (30�XC) temperatures by detecting the secretion of a T6SS substrate, Hcp. This finding suggests that VflT6SS2 may play an important role in the survival of the bacterium in the aquatic environment. The secretion of Hcp is growth phase-dependent and occurs in a narrow range of the growth phase (OD600 from 1.0 to 2.0). Osmolarity also regulates the function of VflT6SS2, as evidenced by our finding that increasing salinity (from 170 to 855 mM of NaCl) and exposure to high osmolarity KCl, sucrose, trehalose, or mannitol (equivalent to 340 mM of NaCl) induced significant secretion of Hcp under growth at 30�XC. Furthermore, we found that although VflT6SS2 was inactive at a higher temperature (37�XC), it became activated at this temperature if higher salinity conditions were present (from 513 to 855 mM of NaCl), indicating that it may be able to function under certain conditions in the infected host. Finally, we showed that the functional expression of VflT6SS2 is associated with anti-bacterial activity. This activity is Hcp-dependent and requires vasH, a transcriptional regulator of T6SS. In sum, our study demonstrates that VflT6SS2 provides V. fluvialis with an enhanced competitive fitness in the marine environment, and its activity is regulated by environmental signals, such as temperature and osmolarity.
( 2015 )
The fur gene as a new phylogenetic marker for Vibrionaceae species identification.
PMID : 25662978 : DOI : 10.1128/AEM.00058-15 PMC : PMC4375339
Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA.
( 2015 )
VibrioBase: A MALDI-TOF MS database for fast identification of Vibrio spp. that are potentially pathogenic in humans.
PMID : 25466918 : DOI : 10.1016/j.syapm.2014.10.009
Mesophilic marine bacteria of the family Vibrionaceae, specifically V. cholerae, V. parahaemolyticus and V. vulnificus, are considered to cause severe illness in humans. Due to climate-change-driven temperature increases, higher Vibrio abundances and infections are predicted for Northern Europe, which in turn necessitates environmental surveillance programs to evaluate this risk. We propose that whole-cell matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiling is a promising tool for the fast and reliable species classification of environmental isolates. Because the reference database does not contain sufficient Vibrio spectra we generated the VibrioBase database in this study. Mass spectrometric data were generated from 997 largely environmental strains and filed in this new database. MALDI-TOF MS clusters were assigned based on the species classification obtained by analysis of partial rpoB (RNA polymerase beta-subunit) sequences. The affiliation of strains to species-specific clusters was consistent in 97% of all cases using both approaches, and the extended VibrioBase generated more specific species identifications with higher matching scores compared to the commercially available database. Therefore, we have made the VibrioBase database freely accessible, which paves the way for detailed risk assessment studies of potentially pathogenic Vibrio spp. from marine environments.
( 2014 )
Evaluation of molecular methods to discriminate the closely related species Vibrio fluvialis and Vibrio furnissii.
PMID : 25242722 : DOI : 10.1016/j.ijmm.2014.09.001
Vibrio furnissii and Vibrio fluvialis are two closely related species which are regarded as emerging human pathogens. Human infections have been mainly associated with consumption of seafood or drinking of contaminated water. V. furnissii strains can be distinguished from V. fluvialis by their ability to produce gas from fermentation of carbohydrates. In this study, we compare two phenotypic (biochemical testing and matrix-assisted laser desorption/ionisation time of flight mass spectrometry, MALDI-TOF MS) and three genotypic techniques (rpoB sequencing, conventional PCR and real-time PCR) for determination of the two species. The methods were evaluated on a collection of 42 V. furnissii and 32 V. fluvialis strains, which were isolated from marine environments and from animals intended for food production. Four of the applied methods allowed the unambiguous discrimination of the two species, while the biochemical testing was the least reliable technique, due to a high variation in the phenotype of gas production from carbohydrates. In view of the One Health concept reliable diagnostic techniques are a prerequisite for preventive public health measurements, as pathogens isolated from animals can cross species borders and methods for detection of sources, reservoirs and ways of transmission of pathogenic bacteria are indispensable for the prevention of infectious diseases in humans and animals.
( 2014 )
Vibrio trends in the ecology of the Venice lagoon.
PMID : 24487545 : DOI : 10.1128/AEM.04133-13 PMC : PMC3993166
Vibrio is a very diverse genus that is responsible for different human and animal diseases. The accurate identification of Vibrio at the species level is important to assess the risks related to public health and diseases caused by aquatic organisms. The ecology of Vibrio spp., together with their genetic background, represents an important key for species discrimination and evolution. Thus, analyses of population structure and ecology association are necessary for reliable characterization of bacteria and to investigate whether bacterial species are going through adaptation processes. In this study, a population of Vibrionaceae was isolated from shellfish of the Venice lagoon and analyzed in depth to study its structure and distribution in the environment. A multilocus sequence analysis (MLSA) was developed on the basis of four housekeeping genes. Both molecular and biochemical approaches were used for species characterization, and the results were compared to assess the consistency of the two methods. In addition, strain ecology and the association between genetic information and environment were investigated through statistical models. The phylogenetic and population analyses achieved good species clustering, while biochemical identification was demonstrated to be imprecise. In addition, this study provided a fine-scale overview of the distribution of Vibrio spp. in the Venice lagoon, and the results highlighted a preferential association of the species toward specific ecological variables. These findings support the use of MLSA for taxonomic studies and demonstrate the need to consider environmental information to obtain broader and more accurate bacterial characterization.
( 2013 )
Quorum sensing regulatory cascades control Vibrio fluvialis pathogenesis.
PMID : 23749976 : DOI : 10.1128/JB.00508-13 PMC : PMC3754567
Quorum sensing (QS) is a process by which individual bacteria are able to communicate with one another, thereby enabling the population as a whole to coordinate gene regulation and subsequent phenotypic outcomes. Communication is accomplished through production and detection of small molecules in the extracellular milieu. In many bacteria, particularly Vibrio species, multiple QS systems result in multiple signals, as well as cross talk between systems. In this study, we identify two QS systems in the halophilic enteric pathogen Vibrio fluvialis: one acyl-homoserine lactone (AHL) based and one CAI-1/AI-2 based. We show that a LuxI homolog, VfqI, primarily produces 3-oxo-C10-HSL, which is sensed by a LuxR homolog, VfqR. VfqR-AHL is required to activate vfqI expression and autorepress vfqR expression. In addition, we have shown that similar to that in V. cholerae and V. harveyi, V. fluvialis produces CAI-1 and AI-2 signal molecules to activate the expression of a V. cholerae HapR homolog through LuxO. Although VfqR-AHL does not regulate hapR expression, HapR can repress vfqR transcription. Furthermore, we found that QS in V. fluvialis positively regulates production of two potential virulence factors, an extracellular protease and hemolysin. QS also affects cytotoxic activity against epithelial tissue cultures. These data suggest that V. fluvialis integrates QS regulatory pathways to play important physiological roles in pathogenesis.
( 2012 )
Vibrio alfacsensis sp. nov., isolated from marine organisms.
PMID : 22286904 : DOI : 10.1099/ijs.0.033191-0
Five strains (CAIM 1831(T), CAIM 1832, CAIM 1833, CAIM 1834 and CAIM 1836) were isolated from cultured sole (Solea senegalensis) in two regions of Spain, two strains (CAIM 404 and CAIM 1294) from wild-caught spotted rose snapper (Lutjanus guttatus) in Mexico, and one strain (CAIM 1835) from corals in Brazil. The 16S rRNA gene sequences of the novel isolates showed similarity to Vibrio ponticus (98.2-98.3%, GenBank accession no. AJ630103) and to a lesser degree to Vibrio furnissii (97.2-97.3%, X76336) and to Vibrio fluvialis (96.9-97.1%, X74703). Multilocus sequence analysis clustered these strains closely together and clearly separated them from phylogenetically related species of the genus Vibrio. Genomic fingerprinting by rep-PCR clustered the novel strains according to their geographical origin. Phenotypic analyses showed a large variation among the new strains, but many tests enabled them to be differentiated from other species of the genus Vibrio. The mean �GT(m) values between the strains analysed here and closely related type strains were above 6.79 �XC. The values between the novel isolates were below 2.35 �XC, well outside the limit suggested for the delineation of a bacterial species. The phenotypic and genotypic data presented here clearly place these new strains as a coherent group within the genus Vibrio, for which we propose the name Vibrio alfacsensis sp. nov. with CAIM 1831(T) (= DSM 24595(T) = S277(T)) as the type strain.
( 2012 )
Vibrio xiamenensis sp. nov., a cellulase-producing bacterium isolated from mangrove soil.
PMID : 22039001 : DOI : 10.1099/ijs.0.033597-0
A taxonomic study was carried out on a cellulase-producing bacterium, strain G21(T), isolated from mangrove soil in Xiamen, Fujian province, China. Cells were Gram-negative, slightly curved rods, motile with a single polar flagellum. The strain grew at 15-40 �XC and in 0.5-10% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain G21(T) belonged to the genus Vibrio and formed a clade with Vibrio furnissii ATCC 350116(T) (97.4% sequence similarity), V. fluvialis LMG 7894(T) (97.1%) and V. ponticus CECT 5869(T) (96.1%). However, multilocus sequence analysis (using rpoA, recA, mreB, gapA, gyrB and pyrH sequences) and DNA-DNA hybridization experiments indicated that the strain was distinct from the closest related Vibrio species. Additionally, strain G21(T) could be differentiated from them phenotypically by the ability to grow in 10% NaCl but not on TCBS plates, its enzyme activity spectrum, citrate utilization, oxidization of various carbon sources, hydrolysis of several substrates and its cellular fatty acid profile. The G+C content of the genomic DNA was 46.0 mol%. The major cellular fatty acids were summed feature 3 (C(16:1)�s7c and/or iso-C(15:0) 2-OH), C(16:0) and C(18:1)�s7c. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol, with trace amounts of diphosphatidylglycerol. The predominant quinones were Q-8 and Q-7. Based on phylogenetic, phenotypic and chemotaxonomic characteristics and DNA-DNA hybridization analysis, it is concluded that strain G21(T) represents a novel species of the genus Vibrio, for which the name Vibrio xiamenensis sp. nov. is proposed. The type strain is G21(T) (= DSM 22851(T) = CGMCC 1.10228(T)).
( 2011 )
Transferable plasmid-mediated quinolone resistance in association with extended-spectrum �]-lactamases and fluoroquinolone-acetylating aminoglycoside-6'-N-acetyltransferase in clinical isolates of Vibrio fluvialis.
PMID : 21683552 : DOI : 10.1016/j.ijantimicag.2011.04.013
Vibrio fluvialis, which causes cholera-like diarrhoea in humans, is one of the aetiological agents of acute diarrhoea in Kolkata, India, and is resistant to many antimicrobial agents. Two V. fluvialis isolates resistant to fluoroquinolones and �]-lactam antimicrobials were found to have mutations in the quinolone resistance-determining regions (QRDRs) of GyrA at position 83 and of ParC at position 85 as well as carrying a 150 kb plasmid harbouring the quinolone resistance gene qnrA1, the ciprofloxacin-modifying enzyme-encoding gene aac(6')-Ib-cr and genes encoding for extended-spectrum �]-lactamases such as bla(SHV) and bla(CTX-M-3). When this large plasmid was transferred to Escherichia coli by conjugation, the transconjugants showed a 10-75-fold increase in the minimum inhibitory concentrations of ciprofloxacin and norfloxacin. The qnrA1 gene was identified in a complex sul1-type integron in a plasmid of the transconjugants. Southern hybridisation and sequence analysis of qnrA1 and its flanking regions confirmed the presence of aac(6')-Ib-cr and bla(CTX-M-3) but these were not associated with the sul1-type integron. Pulsed-field gel electrophoresis (PFGE) revealed that the two V. fluvialis isolates belonged to different clones. Although the presence of many qnr alleles has been reported amongst enteric bacteria in Asian countries, this is the first report on the emergence of qnrA1 in India. qnrA1 along with aac(6')-Ib-cr and bla(CTX-M-3) genes on a mobile plasmid may spread to other bacterial species that are under the selective pressure of fluoroquinolones and �]-lactam antimicrobials in this region.
( 2012 )
Clinical isolates of Vibrio fluvialis from Kolkata, India, obtained during 2006: plasmids, the qnr gene and a mutation in gyrase A as mechanisms of multidrug resistance.
PMID : 22034163 : DOI : 10.1099/jmm.0.037226-0
Resistance profiles and their correlation with genetic factors were investigated in 12 isolates of Vibrio fluvialis obtained from hospitalized patients in Kolkata, India, in 2006. All the strains displayed drug resistance with varying antibiograms. However, resistance to ampicillin and neomycin was common to all of them. Three isolates harboured plasmids carrying drug-resistance genes that could be transferred to recipient strains by conjugation and transformation. PCR results indicated the absence of class 1 integrons and SXT elements in these isolates. A mutation in gyrase A (serine 83��isoleucine) and the presence of the qnrVC-like [corrected] gene were found to contribute towards quinolone resistance. In the 12 isolates, the qnrVC-like [corrected] gene was associated only with two plasmid-bearing isolates, L10734 and L9978, which displayed resistance to quinolones. The gene was transferable during transformation and conjugation, indicating that it was plasmid-borne. Taken together, these data indicate that plasmids, the qnrVC-like [corrected] gene and a mutation in gyrase A were responsible for the observed drug resistance in these strains. To the best of our knowledge, this is the first report of the presence of the qnrVC-like [corrected] allele in V. fluvialis isolates from India.
( 2018 )
Structural dynamics of the transaminase active site revealed by the crystal structure of a co-factor free omega-transaminase from Vibrio fluvialis JS17.
PMID : 30061559 : DOI : 10.1038/s41598-018-29846-0 PMC : PMC6065307
Omega (�s)-transaminase catalyzes the transfer of an amino group from a non-�\ position amino acid, or an amine compound with no carboxylic group, to an amino acceptor, and has been studied intensively because of its high potential utility in industry and pharmatheutics. The �s-transaminase from Vibrio fluvialis JS17 (Vfat) is an amine:pyruvate transaminase capable of the stereo-selective transamination of arylic chiral amines. This enzyme exhibits extraordinary enantio-selectivity, and has a rapid reaction rate for chiral amine substrates. In this study, we report the crystal structure of the apo form of Vfat. The overall structure of Vfat was typical of other class III aminotransferase exhibiting an N-terminal helical domain, a small domain, and a large domain. Interestingly, the two subunits of apo Vfat in the asymmetric unit had different structures. A comparison of the overall structure to other transaminases, revealed that the structures of the N-terminal helical domain and the large domain can be affected by cofactor occupancy, but the structural rearrangement in these regions can occur independently.
( 2018 )
A Highly Promiscuous Integron, Plasmids, Extended Spectrum Beta Lactamases and Efflux Pumps as Factors Governing Multidrug Resistance in a Highly Drug Resistant Vibrio fluvialis Isolate BD146 from Kolkata, India.
PMID : 29434398 : DOI : 10.1007/s12088-017-0687-8 PMC : PMC5801176
In an earlier study from this laboratory, Vibrio fluvialis BD146, a clinical isolate from Kolkata, India, 2002, was found to be resistant to all the fourteen antibiotics tested. It harboured a high copy number plasmid pBD146 and a low copy number plasmid. In the present study, a more detailed analysis was carried out to unravel different resistance mechanisms in this isolate. Sequencing showed that variable region of class 1 integron located on low copy number plasmid harbored arr3-cmlA-blaOXA10-aadA1 gene cassettes. Analysis for extended spectrum beta lactamases (ESBLs) revealed that BD146 was ESBL positive. Efflux pumps were involved in the drug resistance phenotype for chloramphenicol, kanamycin, streptomycin and tetracycline. Sequence analysis of pBD146 revealed the presence of genes encoding BDint an integrase with a unique sequence having little similarity to other known integrases, toxin-antitoxin (parE/parD), a replicase, trimethoprim resistance (dfrVI) and quinolone resistance (qnrVC5). Presence of cmlA, putative novel integrase and toxin-antitoxin system in V. fluvialis has been documented for the first time in this report. pBD146 showed 99% sequence similarity with pVN84 from V. cholerae O1 of Vietnam, 2004 and a plasmid from V. parahaemolyticus v110 of Hong Kong, 2010. Conjugation experiments proved the ability of pBD146 and the low copy number plasmid, to get transferred to another host imparting their antibiotic resistance traits to the transconjugants. Therefore, present study has indicated that plasmids played an important role for dissemination of drug resistance.
( 2013 )
The virulence phenotypes and molecular epidemiological characteristics of Vibrio fluvialis in China.
PMID : 23522652 : DOI : 10.1186/1757-4749-5-6 PMC : PMC3636005
Vibrio fluvialis is considered to be an emerging foodborne pathogen and has been becoming a high human public health hazard all over the world, especially in coastal areas of developing countries and regions with poor sanitation. The distribution of virulence factors, microbiological and molecular epidemiological features of V. fluvialis isolates in China remains to be examined. PCR targeted at the virulence determinants and phenotype tests including metabolism, virulence and antibiotic susceptibility were performed. Pulsed-field gel electrophoresis (PFGE) analysis was used to access the relatedness of isolates. A strain with deletion of the arginine dihydrolase system was first reported and proved in molecular level by PCR. Virulence genes vfh, hupO and vfpA were detected in all strains, the ability to produce hemolysin, cytotxin, protease and biofilm formation varied with strains. High resistance rate to �]-lactams, azithromycin and sulfamethoxazole were observed. Twenty-seven percent of test strains showed resistant to two and three antibiotics. PFGE analysis demonstrated great genetic heterogeneity of test V. fluvialis strains. This study evaluated firstly the biological characteristics and molecular epidemiological features of V. fluvialis in China. Some uncommon biochemical characteristics were found. Virulence genes were widely distributed in the isolates from patient and seafood sources, and the occurrence of virulence phenotypes varied with strains. Continued and enhanced laboratory based-surveillance is needed in the future together with systematically collection of the epidemiological information of the cases or the outbreaks.
( 2013 )
Comparative analysis of mobilizable genomic islands.
PMID : 23204461 : DOI : 10.1128/JB.01985-12 PMC : PMC3554006
Mobilizable genomic islands (MGIs) are small genomic islands of less than 35 kbp containing an integrase gene and a sequence that resembles the origin of transfer (oriT) of an integrating conjugative element (ICE). MGIs have been shown to site-specifically integrate and excise from the chromosome of bacterial hosts and hijack the conjugative machinery of a coresident ICE to disseminate. To date, MGIs have been described in three strains belonging to three different Vibrio species. In this study, we report the discovery of 11 additional putative MGIs found in various species of Vibrio, Alteromonas, Pseudoalteromonas, and Methylophaga. We designed an MGI capture system that allowed us to relocate chromosomal MGIs onto a low-copy-number plasmid and facilitate their isolation and sequencing. Comparative genomics and phylogenetic analyses of these mobile genetic elements revealed their mosaic structure and their evolution through recombination and acquisition of exogenous DNA. MGIs were found to belong to a larger family of genomic islands (GIs) sharing a similar integrase gene and often integrated into the same integration site yet exhibiting a different mechanism of regulation of excision and mobilization. We found that MGIs can excise only when an ICE of the SXT/R391 family is coresident in the same cell, while GIs still excise regardless.
( 2013 )
Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin.
PMID : 23012440 : DOI : 10.1093/protein/gzs065
Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related �]-ketoesters.
( 2012 )
Dynamics of the SetCD-regulated integration and excision of genomic islands mobilized by integrating conjugative elements of the SXT/R391 family.
PMID : 22923590 : DOI : 10.1128/JB.01093-12 PMC : PMC3486078
Mobilizable genomic islands (MGIs) are small genomic islands that are mobilizable by SXT/R391 integrating conjugative elements (ICEs) due to similar origins of transfer. Their site-specific integration and excision are catalyzed by the integrase that they encode, but their conjugative transfer entirely depends upon the conjugative machinery of SXT/R391 ICEs. In this study, we report the mechanisms that control the excision and integration processes of MGIs. We found that while the MGI-encoded integrase Int(MGI) is sufficient to promote MGI integration, efficient excision from the host chromosome requires the combined action of Int(MGI) and of a novel recombination directionality factor, RdfM. We determined that the genes encoding these proteins are activated by SetCD, the main transcriptional activators of SXT/R391 ICEs. Although they share the same regulators, we found that unlike rdfM, int(MGI) has a basal level of expression in the absence of SetCD. These findings explain how an MGI can integrate into the chromosome of a new host in the absence of a coresident ICE and shed new light on the cross talk that can occur between mobilizable and mobilizing elements that mobilize them, helping us to understand part of the rules that dictate horizontal transfer mechanisms.